Modified ethylenediamine-n,n&#39;-disuccinate:ethylenediamine lyase

ABSTRACT

The present invention provides a modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase. The present invention also provides a protein that comprises the amino acid sequence represented by SEQ ID NO: 1; or a protein that comprises an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 1 by deletion, substitution, or addition of one or more amino acid residues, and has an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.

CROSS REFERENCE TO PRIOR APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/569,339,filed Jan. 10, 2007, which is a U.S. national phase application under 35U.S.C. §371 of International Application No. PCT/JP2004/007226 filed May20, 2004. The International Application was published in Japanese onDec. 1, 2005 as International Publication No. WO 2005/113766 under PCTArticle 21(2). All of these prior applications are hereby incorporatedby reference in their entireties.

TECHNICAL FIELD

The present invention relates to a novel protein having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity and agene encoding such protein. Further, the present invention relates to amodified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase that canbe derived as a mutant of the enzyme; a gene encoding the modifiedethylenediamine-N,N′-disuccinate:ethylenediamine lyase; a recombinantDNA containing a gene DNA encoding the modifiedethylenediamine-N,N′-disuccinate:ethylenediamine lyase; a transformantor transductant bearing the recombinant DNA containing the gene encodingthe modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase; anda method for producing diaminoalkylene-N,N′-disuccinates by using such atransformant or transductant.

BACKGROUND

Diaminoalkylene-N,N′-disuccinates are important as intermediates ofsynthesis of medicines and agricultural chemicals, and have a uniqueproperty to capture heavy metals. Therefore, optically active forms ofsuch compounds, which may be susceptible to biodegradation once releasedin nature, are expected to be potentially useful as chelating agents,builders for detergents, etc.

Previously, the present inventors proposed a novel method forefficiently preparing optically activeS,S-diaminoalkylene-N,N′-disuccinates from fumaric or maleic acid, andvarious amines by utilizing the catalytic action of the microorganism[JP Patent Publication (Kokai) Nos. 9-140390A (1997), 9-289895A (1997),and 10-52292A (1998)]. Further, we were successful in increasing acatalytic activity of the microorganism and improving a productivity ofthe microorganism by isolating and identifying anethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene followed bygenetic recombination [JP Patent Publication (Kokai) No. 10-210984A(1998)].

In general, it is well known that a fumarase is present in microbialcells. Fumarase is an enzyme used to produce malic acid by adding waterto fumaric acid. Therefore, when diaminoalkylene-N,N′-disuccinates areprepared by using microorganisms having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity, afumarase in the microbial cells needs to be inactivated for preventinggeneration of by-products such as malic acid and the like. In order todo so, the present inventors previously found that when the microbialcells are treated in an alkaline aqueous solution, a fumarase activityin the cells can be reduced [JP Patent Publication (Kokai) No.11-196882A (1999)].

In the above method for inactivating a fumarase, the inactivation rateof fumarase depends on the treatment temperature. Thus, the higher isthe treatment temperature, the faster the inactivation. Moreover, thestability of fumarase varies with host microorganisms, but even when amicroorganism whose fumarase is not inactivated readily is used, ahigher treatment temperature is preferred. Accordingly, the objective ofthe present invention is to provide anethylenediamine-N,N′-disuccinate:ethylenediamine lyase having improvedheat resistance.

SUMMARY OF THE INVENTION

Vigorous investigation for attaining the above objective by the presentinventors has led to the discovery that in the amino acid sequence ofethylenediamine-N,N′-disuccinate:ethylenediamine lyase derived fromnovel Brevundimonas diminuta strain MR-E001, by substituting at leastone or more amino acid residues with residues selected from the group ofnatural amino acids, heat resistance of the enzyme is improved. Thereby,the present invention has been completed. Specifically, the presentinvention includes:

(1) A protein comprising an amino acid sequence described in SEQ ID NO:1, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseactivity.(2) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by deletion, substitution, or addition ofone or more amino acid residues, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(3) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least one amino acidresidue of a lysine residue at 120, an isoleucine residue at 166, and analanine residue at 365 with a different amino acid residue, and havingan ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(4) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least a lysineresidue at 120 with glutamic acid, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(5) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least the isoleucineresidue at 166 with serine, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(6) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least an isoleucineresidue at 166 with threonine, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(7) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least an alanineresidue at 365 with valine, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(8) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least a lysineresidue at 120 with glutamic acid and an isoleucine residue at 166 withserine, and having an ethylenediamine-N,N′-disuccinate:ethylenediaminelyase activity.(9) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least a lysineresidue at 120 with glutamic acid and an isoleucine residue at 166 withthreonine, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(10) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least an isoleucineresidue at 166 with serine and an alanine residue at 365 with valine,and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseactivity.(11) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least an isoleucineresidue at 166 with threonine and an alanine residue at 365 with valine,and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseactivity.(12) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least a lysineresidue at 120 with glutamic acid and an isoleucine residue at 166 withserine an alanine residue at 365 with valine, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(13) A protein comprising an amino acid sequence derived from the aminoacid sequence of SEQ ID NO: 1 by substitution of at least a lysineresidue at 120 with glutamic acid and an isoleucine residue at 166 withthreonine and an alanine residue at 365 with valine, and having anethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.(14) A gene encoding the protein according to (1).(15) A gene of the following (a) or (b):(a) a gene comprising a nucleotide sequence described in SEQ ID NO: 2;or(b) a gene that hybridizes under stringent conditions to a DNAcomprising a sequence complementary to a DNA comprising the nucleotidesequence of SEQ ID NO: 2 or a portion thereof, and encodes a proteinhaving an ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseactivity.(16) A gene encoding the protein according to (2).(17) The gene according to (16) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by deletion, substitutionor addition of one or more bases.(18) A gene encoding the protein according to (3).(19) The gene according to (18) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of at leastone base of bases from 358 to 360, from 496 to 498, and from 1093 to1095 with a different base.(20) A gene encoding the protein according to (4).(21) The gene according to (20) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesAAA from 358 to 360 with GAA or GAG.(22) The gene according to (20) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,adenine, at 358 with guanine.(23) A gene encoding the protein according to (5).(24) The gene according to (23) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T).(25) The gene according to (23) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of thebase, thymine, at 497 with guanine.(26) A gene encoding the protein according to (6).(27) The gene according to (26) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesATC from 496 to 498 with ACN (N refers to A, G, C or T).(28) The gene according to (26) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,thymine, at 497 with cytosine.(29) A gene encoding the protein according to (7).(30) The gene according to (29) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesGCC from 1093 to 1095 with GTN (N refers to A, G, C or T).(31) The gene according to (29) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,cytosine, at 1094 with thymine(32) A gene encoding the protein according to (8).(33) The gene according to (32) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesAAA from 358 to 360 with GAA or GAG, and bases ATC from 496 to 498 withAGC, AGT, or TCN (N refers to A, G, C or T).(34) The gene according to (32) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,adenine, at 358 with guanine, and a base, thymine, at 497 with guanine.(35) A gene encoding the protein according to (9).(36) The gene according to (35) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesAAA from 358 to 360 with GAA or GAG, and bases ATC from 496 to 498 withACN (N refers to A, G, C or T), respectively.(37) The gene according to (35) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,adenine, at 358 with guanine, and a base, thymine, at 497 with cytosine,respectively.(38) A gene encoding the protein according to (10).(39) The gene according to (38) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T),and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T),respectively.(40) The gene according to (38) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,thymine, at 497 with guanine, and a base, cytosine, at 1094 withthymine, respectively.(41) A gene encoding the protein according to (11).(42) The gene according to (41) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesATC from 496 to 498 with ACN (N refers to A, G, C or T), and bases GCCfrom 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.(43) The gene according to (41) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,thymine, at 497 with cytosine, and a base, cytosine, at 1094 withthymine, respectively.(44) A gene encoding the protein according to (12).(45) The gene according to (44) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesAAA from 358 to 360 with GAA or GAG, bases ATC from 496 to 498 with AGC,AGT, or TCN (N refers to A, G, C or T), and bases GCC from 1093 to 1095with GTN (N refers to A, G, C or T), respectively.(46) The gene according to (44) comprising a nucleotide sequence whereinthe base, adenine, at 358 is substituted with guanine, the base,thymine, at 497 with guanine, and the base, cytosine, at 1094 withthymine in the nucleotide sequence of SEQ ID NO: 2.(47) A gene encoding the protein according to (13).(48) The gene according to (47) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of basesAAA from 358 to 360 with GAA or GAG, bases ATC from 496 to 498 with ACN(N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (Nrefers to A, G, C or T), respectively.49) The gene according to (47) comprising a nucleotide sequence derivedfrom the nucleotide sequence of SEQ ID NO: 2 by substitution of a base,adenine, at 358 with guanine, a base, thymine, at 497 with cytosine, anda base, cytosine, at 1094 with thymine, respectively.(50) A recombinant wherein the gene DNA according to any one of (14) to(49) is inserted into a DNA vector.(51) A transformant or transductant comprising the recombinant DNAaccording to (50).(52) A method of producing anethylenediamine-N,N′-disuccinate:ethylenediamine lyase, comprising astep of culturing the transformant or transductant according to (51) tocollect the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase fromthe resulting culture.(53) A method of producing a diaminoalkylene-N,N′-disuccinate,comprising a step of reacting fumaric acid and a diamine in the presenceof the transformant or transductant according to (51) to collect thediaminoalkylene-N,N′-disuccinate from the resulting reaction products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of the plasmid pEDS9001.

FIG. 2 is a restriction map of the plasmid pEDS9003.

FIG. 3 is a restriction map of the plasmid pEDS9020.

FIG. 4 is an assembly scheme of the expression vector pFY529V.

FIG. 5 is a graph for evaluating heat resistance of the single mutants.

FIG. 6 is a schematic representation to illustrate the constructions ofthe single mutants and the multiple mutants: square indicates themutations of T497C (Ile166Thr), triangle indicates the mutations ofA358G (Lys120Glu), circle indicate the mutations of T497G (Ile166Ser),and diamond indicates the mutations of C1094T (Ala365Val), respectively.

FIG. 7 is a graph for evaluating heat resistance of the multiplemutants.

DETAILED DESCRIPTION

The present invention is described in detail below. In the presentinvention, the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase(also named “EDDSase”) refers to an enzyme capable of reversiblyproducing ethylenediamine-N,N′-disuccinate from fumaric acid andethylenediamine, but it may produce ethylenediamine-N-monosuccinic aciddepending on reaction conditions. Further, the present enzyme alsoreacts on other diamines in addition to ethylenediamine, producingcorresponding diaminoalkylene-N,N′-disuccinates. Further, many ofresulting diaminoalkylene-N,N′-disuccinates are optically active forms,but some generated by the enzyme are racemic compounds. A group ofenzymes exhibiting such reactivity have been found in bacteria belongingto plural genera that have been isolated and identified from nature bythe present inventors. These bacteria are described in the above JPPatent Publication (Kokai) Nos. 9-140390A (1997), 9-289895A (1997), and10-52292A (1998). Moreover, the present applicants were successful notonly in isolating from Brevundimonas sp. strain TN-3 described in PatentPublication (Kokai) No. 10-52292A (1998) anethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene to spell outthe amino acid sequence and the gene sequence thereof for the firsttime, but also in forming transformants that potentially can express thegene product thereof in large quantities [JP Patent Publication (Kokai)No. 10-210984A (1998)].

On the other hand, recent advancement in recombinant DNA technologymakes it possible to form variants wherein one or more amino acids thatconstitute an enzyme are lost, added, deleted, inserted, or substitutedwith other amino acids, without essentially changing the action of suchan enzyme. It is known that compared with the wild type enzyme withoutmutation, depending on the locations of amino acid residues substituted,lost, added, deleted, or inserted as well as types of amino acidssubstituted, these variants may exhibit significantly improvedproperties such as resistance to organic solvents, heat resistance, acidresistance, alkali resistance, substrate specificity, and substrateaffinity. These improved properties may result in more stable enzymes ascatalysts, simpler reaction processes, increased reaction yields, etc.,and thereby may contribute to great reduction in production cost inmanufacturing where enzyme reactions are utilized. Therefore, attemptsare made to improve various properties in a large number of enzymes todevelop useful modified enzymes.

Herein, “wild type” means that an amino acid sequence that constitutesan enzyme kept in a microorganism separated from nature, and anucleotide sequence encoding such an enzyme are, either intentionally orunintentionally, not lost, deleted, inserted, or substituted with otheramino acids or bases.

The present inventors conducted screening of microorganisms having suchan enzyme activity for further usefulethylenediamine-N,N′-disuccinate:ethylenediamine lyase. As a result,Brevundimonas diminuta strain MR-E001 (hereinafter also referred to theMR-E001 strain), which has a high activity of the enzyme, was isolated,and an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene wasobtained from the MR-E001 strain. In addition, it was discovered that inthe amino acid sequence of the enzyme, by substituting at least one ormore amino acid residues with residues selected from the group ofnatural amino acids, heat resistance of the enzyme is improved. Thereby,the present invention has been completed.

The modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase ofthe present invention can be obtained, for example, by the followingmethod. First, from the MR-E001 strain, a wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene is obtained.For obtaining the gene, any known technique can be used. For example,chromosomal DNA prepared from the MR-E001 strain is used as a templateand PCR (Polymerase Chain Reaction) is conducted to obtain a DNAfragment containing a part of the gene. For the primers used for PCR, ingeneral, degenerate primers can be used, which are designed based on theamino acid sequence obtained by amino acid analysis after isolation andpurification of the enzyme. Further, when the sequence of the gene ofinterest is expected to be homologous with a sequence of the genederived from a different species that has been already known, or when agene homologous with a sequence of the gene derived from a differentspecies that has been already known is to be obtained, degenerateprimers can be designed for PCR according to the amino acid sequenceinformation encoded by such a known gene derived from the differentspecies. By using the primers designed thereby, the chromosomal DNA fromthe MR-E001 strain is used as a template and PCR is conducted, and theresulting amplified DNA product is used as a probe for colonyhybridization that will be conducted later.

Next, a DNA library is prepared. The chromosome from the MR-E001 strainprepared according to a known method, for example, the method made bySaito and Miura [Biochem. Biophys. Acta, 72, 619 (1963)], is cleaved orpartially cleaved by a suitable restriction enzyme, which is thenligated to a vector DNA that has been treated by a restriction enzymethat can generate cleaved terminals linkable to such cleaved terminalsof the restriction enzyme described above, so that transformants ortransductants of a suitable microorganism host are formed, and therebythe DNA library of the chromosome is produced. Examples of themicroorganisms that can be hosts for the transformants or thetransductants include, when Escherichia coli is used, E. coli strainK12, strain JM109, strain XL1-Blue, etc., but should not be particularlylimited to these. Examples of the plasmid DNA used for forming thetransformants include, when E. coli is the host, pBR322, pUC18,pBluescript II SK(+), etc., which have auto-replicable regions in E.coli. Further, the vector DNA should not be limited to the above plasmidvector DNAs, but phage vector DNAs may be used to form transductants.

Herein, in describing gene manipulation processes, microorganismscontaining a recombinant DNA of interest are referred to astransformants when plasmid vector DNAs are used, and transductants whenphage vector DNAs are used. All of the above transformants andtransductants are included in the present invention. Examples, whereintransformants are created utilizing plasmid vector DNAs, are describedbelow.

Colony hybridization is conducted for the resulting DNA library of thechromosome, using the PCR-amplified DNA product described above as aprobe. The colony hybridization may be an ordinary method. For example,the one described below can be used. Specifically, transformants fromthe chromosomal DNA library grown on an agar medium are transferred to anylon membrane, and then the DNA is fixed by cytolysis. The amplifiedDNA product by PCR described above is rendered to be a probe bylabeling, for example, using a DNA Labeling kit (from RocheDiagnostics), and then hybridized with the membrane. Positive clones canbe selected, for example, by using a DNA Luminescent Detection kit (fromRoche Diagnostics). From the resulting positive clones, plasmid DNA isprepared according to the ordinary method, optionally subcloning isconducted, and then nucleotide sequences of the inserted fragments aredetermined Any method can be used for the determination of thenucleotide sequences, and usually the nucleotide sequences can bedetermined by the dideoxy method (Methods in Enzymology, 101, 20-78,1983) using a commercial kit or the like. Thus, the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene derived fromthe MR-E001 strain can be obtained, and also an amino acid sequence anda nucleotide sequence thereof can be determined.

Next, for obtaining a modifiedethylenediamine-N,N′-disuccinate:ethylenediamine lyase, wherein in theamino acid sequence of the resulting wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase, at least oneamino acid residue is substituted with a different natural amino acidresidue, any method can be used and usually a well known method can beused. Specifically, examples of methods for treating the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNA include:to contact and react a mutagenic chemical such as hydroxylamine andnitrous acid; to induce mutations by irradiating ultraviolet rays; toinduce mutations on a random basis by using PCR; to generatesite-specific substitutions by utilizing a commercial kit; toselectively cleave gene DNA and then remove and add selectedoligonucleotides for ligation; and the like.

After an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNAhaving a distinct mutation caused by any one of the above treatments isproduced, transformants is formed. As the plasmid vectors that can beused for transformation, for example, when E. coli is the host, thoseplasmid vectors included in the step for preparing the DNA librarydescribed above may be used, but it is preferable to use an expressionvector with high expression efficiency, such as an expression vector,pKK233-2 (from Amersham), which has a trc promoter, or a derivative ofpKK233-2, i.e., pFY529V, which will be described later in an example, inorder to efficiently detect a remaining activity ofethylenediamine-N,N′-disuccinate:ethylenediamine lyase after heattreatment in the later step for screening.

However, the vectors and the hosts used in the present invention shouldnot be limited to those plasmids described above and E. coli. Examplesof the vectors include plasmid DNA, bacteriophage DNA, retrotransposonDNA, artificial chromosomal DNA, etc.

The ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNA ofthe present invention requires genes to be incorporated into a vector,so that the gene DNA can be expressed in a host thereof. Therefore, tothe vector of the present invention, in addition to the gene DNA of thepresent invention, a promoter, a terminator, an enhancer, a splicingsignal, a poly-A addition signal, a selection marker, a ribosome-bindingsequence (SD sequence), etc., can be linked. Further, examples of theselection markers include a dihydrofolate reductase gene, an ampicillinresistance gene, a neomycin resistance gene, etc.

The transformant of the present invention can be obtained bytransferring the recombinant vector of the present invention into a hostin a manner that the ethylenediamine-N,N′-disuccinate:ethylenediaminelyase gene can be expressed. Examples of the hosts include bacteria suchas E. coli, Bacillus subtilis, etc. In addition, yeast, animal cells,insect cells, plant cells, etc., can be also used.

Examples of bacteria from the genus of Escherichia include Escherichiacoli, etc., and examples of bacteria from the genus of Bacillus includeBacillus subtilis, etc. There should be no particular restriction on howrecombinant vectors are transferred into bacteria, and any technique fortransferring DNA into bacteria can be used. Examples of such techniquesinclude: to utilize calcium ions; electroporation; and the like.

When yeast is the host, for example, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris, etc., are used. There shouldbe no particular restriction on how recombinant vectors are transferredinto yeast, and any technique for transferring DNA into yeast can beused. Examples of such techniques include: electroporation; thespheroplast method; the lithium acetate method; and the like.

When animal cells are the host, monkey COS-7 cells, Vero cells, CHOcells; mouse L cells; rat GH3 cells; human FL cells; and the like areused. Examples of techniques for transferring recombinant vectors intoanimal cells include: electroporation; the Ca-phosphate method;lipofection; and the like.

When insect cells are the host, Sf9 cells, Sf21 cells, and the like areused. Examples of techniques for transferring recombinant vectors intoinsect cells include: the Ca-phosphate method; lipofection;electroporation; and the like.

When plant cells are the host, examples include corn, rice, tobacco, andthe like, but should not be limited to these. Examples of technique fortransferring recombinant vectors into plant cells include: theAgrobacterium method; the particle gun method; the PEG method;electroporation; and the like.

As described above, various transformants that bear recombinant DNAcontaining the ethylenediamine-N,N′-disuccinate:ethylenediamine lyasegene can be provided.

When a host is E. coli, the resulting transformants are cultured on anagar medium to form colonies followed by liquid culture to produce anethylenediamine-N,N′-disuccinate:ethylenediamine lyase. The resultingculture, for example, is subjected to heat treatment for 30 min. at 40to 65° C. followed by the determination of remainingethylenediamine-N,N′-disuccinate:ethylenediamine lyase activities. Atransformant with a higher remainingethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity isselected. A nucleotide sequence of theethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene insertedinto the recombinant DNA of the resulting superior transformant therebycan be determined, for example, by the dideoxy method.

The nucleotide sequence of theethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNA of thepresent invention is represented by SEQ ID NO: 2, and an amino acidsequence encoded by the gene of the present invention is represented bySEQ ID NO: 1.

Further, it may be possible to develop a mutated enzyme whose heatresistance is more improved than a single mutant by combining pluraldifferent single-substitution mutations, each of which provides heatresistance to the enzyme, to form a multiple mutant. Any technique canbe used for forming a multiple mutant, and examples include: to generatea site-specific substitution by using an artificial single-strandoligonucleotide; to cleave a DNA fragment containing multiple differentsingle mutated sites by restriction enzymes, which are then ligated; andthe like.

Therefore, as long as theethylenediamine-N,N′-disuccinate:ethylenediamine lyase has its activity,mutation by deletion, substitution, addition, and the like can arise ata plurality of, preferably one or some, amino acids in the amino acidsequence of SEQ ID NO: 1. For example, from 1 to 10, preferably from 1to 5, amino acids may be deleted from the amino acid sequence of SEQ IDNO: 1; from 1 to 10, preferably from 1 to 5, amino acids may be added tothe amino acid sequence of SEQ ID NO: 1; or from 1 to 10, preferablyfrom 1 to 5, amino acids in the amino acid sequence of SEQ ID NO: 1 maybe substituted with other amino acids.

Particularly, in the present invention, it is preferable that at leastone amino acid of Lys at 120, Ile at 166, and Ala at 365 is substitutedby a different amino acid in the amino acid sequence of SEQ ID NO: 1.The amino acid substitutions at the above three locations can beoptionally combined. Aspects of preferred substitutions are shown below,wherein in the following aspects of substitutions, numerals refer tolocation numbers in the amino acid sequence of SEQ ID NO: 1; letters inthe left side of the numbers refer to amino acids (in single letters)before substitution; and letters in the right side of the numbers referto amino acids (in single letters) after substitution.

K120E I166S I166T A365V (K120E, I166S) (K120E, I166T) (I166S, A365V)(I166T, A365V) (K120E, I166S, A365V) (K120E, I166T, A365V)

Moreover, a base substitution so as to generate the substitution ofK120E is described below.

The locations in the nucleotide sequence of SEQ ID NO: 2 correspondingto Lys at 120 are from 358 to 360, and the nucleotide sequence is “AAA.”On the other hand, the codon for glutamic acid is GAA or GAG. Hence, inthe present invention, bases can be substituted so that AAA becomes GAAor GAG. In particular, it is preferable that A at 358 is substitutedwith G (AAA→GAA).

Similarly to the above, so as to generate the substitution of I166S, inthe nucleotide sequence of SEQ ID NO: 2, the bases ATC from 496 to 498can be substituted with AGC, AGT, ACA, ACC, ACG, or ACT. In particular,it is preferable that T at 497 is substituted with G (ATC→AGC). So as togenerate the substitution of I166T, in the nucleotide sequence of SEQ IDNO: 2, the bases ATC from 496 to 498 can be substituted with ACA, ACC,ACG, or ACT. In particular, it is preferable that T at 497 issubstituted with C (ATC→ACC).

So as to generate the substitution of A365V, in the nucleotide sequenceof SEQ ID NO: 2, the bases GCC from 1093 to 1095 can be substituted withGTA, GTG, GTC, or GTT. In particular, it is preferable that C at 1094 issubstituted with T (GCC→GTC).

However, in the present invention, the amino acids after substitutionshould not be limited to the above examples. Thus, so as that at leastone amino acid residue at 120, 166, or 365 in the amino acid sequence ofSEQ ID NO: 1 is substituted with a different amino acid from theexamples above, at least one base of the codons encoding such aminoacids can be substituted with another base.

Herein, “ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseactivity” refers to a catalytic activity to produce adiaminoalkylene-N,N′-disuccinate by reacting fumaric acid and a diamine

Furthermore, a gene that hybridizes under stringent conditions to DNAmade of a sequence complementary to the DNA made of the nucleotidesequence of SEQ ID NO: 2 or a part of such a sequence, and also encodesa protein having the ethylenediamine-N,N′-disuccinate:ethylenediaminelyase activity is included in the gene of the present invention. Thestringent conditions refer to typical conditions in which specifichybridization occurs. Examples include conditions where highlyhomologous nucleic acids, i.e., DNAs with 80% or more, preferably 90% ormore, homology, also each DNA encoding a protein having theethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity,hybridize each other, whereas DNAs with less homology do no hybridizeeach other. More specifically, it refers to conditions in which thesodium concentration is from 300 to 2000 mM, preferably from 600 to 900mM, and the temperature is from 40 to 75° C., preferably from 55 to 65°C.

Once the nucleotide sequence of the present invention is determined, thegene DNA of the present invention can be thereafter obtained by PCR,wherein chemically synthesized or cloned DNA is a template, or byhybridization using a DNA fragment having such a nucleotide sequence asa probe.

In the present invention, the heat resistance refers to a propertycapable of retaining the enzyme activity even in the temperature rangewhere the wild type ethylenediamine-N,N′-disuccinate:ethylenediaminelyase is deactivated. Such a temperature range is from 45 to 60° C.,preferably from 50 to 60° C. In the range of from 50 to 60° C., themodified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase canretain 60% of the enzyme activity exhibited at 50° C.

The resulting transformant bearing the recombinant plasmid DNAcontaining the modified ethylenediamine-N,N′-disuccinate:ethylenediaminelyase gene wherein a single mutation or multiple mutations aretransferred can be used to produce anethylenediamine-N,N′-disuccinate:ethylenediamine lyase. In addition, theabove transformant (e.g., an E. coli transformant) can be used toproduce diaminoalkylene-N,N′-succinic acids.

For producing the ethylenediamine-N,N′-disuccinate:ethylenediaminelyase, the above transformant can be cultured to collect theethylenediamine-N,N′-disuccinate:ethylenediamine lyase from such aculture. The term “culture” refers to any of culture supernatant,cultured cells or cultured cell mass; or destructed cells or cell mass.For culturing the transformant of the present invention, an ordinarymethod used for culturing a host is followed.

For a medium for culturing a transformant obtained from a bacterial hostsuch as E. coli or yeast, any medium, natural or synthetic, containing acarbon source, a nitrogen source, inorganic salts, etc., that can beutilized by the microorganism and capable of efficiently culturing thetransformant can be used. Examples of carbon sources includecarbohydrates such as glucose, fructose, sucrose, starch; organic acidssuch as acetic acid, propionic acid; and alcohols such as ethanol,propanol. Examples of nitrogen sources include ammonia; ammonium saltsof inorganic and organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate, and ammonium phosphate, and othernitrogen-containing compounds, as well as peptone, meat extract, cornsteep liquor, and the like. Examples of inorganic salts includepotassium dihydrogenphosphate, potassium hydrogenphosphate, magnesiumphosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, copper sulfate, calcium carbonate. The culture isusually performed under aerobic conditions such as shake culture, orculture by aeration with stirring. With a solution containing aninorganic or organic acid, or an alkali, the pH is adjusted. During theculture, an antibiotic such as ampicillin or tetracycline may beoptionally added to the medium.

When a microorganism transformed with an expression vector using apromoter is cultured and the promoter is inductive, an inducer may beoptionally added to the medium. For example, when a microorganismtransformed with an expression vector having a promoter that isinducible with isopropyl-β-D-thiogalactoside (IPTG) is cultured, IPTG,etc., can be added to the medium. Further, when a microorganismtransformed with an expression vector using a trp promoter that isinducible with indoleacetic acid (IAA) is cultured, IAA, etc., can beadded to the medium.

Examples of media for culturing a transformant obtained from an animalcell host include media such as general ones, e.g., a RPMI1640 mediumand a DMEM medium; or a medium wherein fetal bovine serum, etc., areadded to a RPMI1640 or DMEM medium. The culture is usually performedunder 5% CO₂ at 37° C. for from 1 to 30 days. During the culture, anantibiotic such as kanamycin or penicillin may be optionally added tothe medium.

When the protein of the present invention is produced within the cellmass or cells following the culture, the protein of interest iscollected by destructing the cell mass or the cells by ultrasonic,repeated freeze and thawing, homogenizing, etc. Further, when theprotein of the present invention is produced outside a microorganism orcells, the culture solution per se is used, or the microorganism or thecells are removed by centrifugation or the like. Then, the protein ofthe present invention can be isolated and purified by an ordinarybiochemical technique used for protein isolation and purification.Examples of such techniques include ammonium sulfate precipitation, gelchromatography, ion-exchange chromatography, and affinitychromatography, which may be used alone or in any proper combinationthereof.

When a transformant is a plant cell or plant tissue, the culture can beperformed by using an ordinary plant culture medium, for example, an MSbasal medium, LS basal medium, etc. Any ordinary culture method, eitherliquid culture or solid culture, can be used.

For purifying the protein of the present invention from the culture,cells are first destructed by cytolysis using an enzyme such ascellulase and pectinase, ultrasonic destruction, grinding, and the like.Then, insoluble matters are removed by filtration or centrifugation toobtain a crude protein solution. The protein of the present invention ispurified from the above crude solution by salt precipitation,chromatography of various types (e.g., gel filtration chromatography,ion-exchange chromatography, affinity chromatography, etc.),SDS-polyacrylamide gel electrophoresis, and the like, or optionally thecombination thereof.

The production of the diaminoalkylene-N,N′-disuccinate is describedbelow. An ordinary solid culture may be used for culturing an E. colitransformant, but it is preferable to use liquid culture as much aspossible. A medium for the culture, for example, containing one or morenitrogen sources such as yeast extract, tryptone, polypeptone, cornsteep liquor, percolate of soy bean and wheat bran loaded with one ormore inorganic salts such as sodium chloride, potassiumdihydrogenphosphate, potassium hydrogenphosphate, magnesium sulfate,magnesium chloride, ferric chloride, ferric sulfate, and manganesesulfate, and optionally further with sugar materials, vitamins, etc., isused. In addition, the initial pH of the medium is suitably adjustedfrom 7 to 9. Preferably, submerged culture, shake culture, stationeryculture, or the like is performed from 25 to 42° C. for from 6 to 24hrs.

After the completion of the culture, the resulting microbial cells areharvested, washed with a suitable buffer solution, for example, a 50 mMboric acid buffer solution (pH 9.0), and then suspended in the buffersolution to prepare a cell suspension. For example, by heating this cellsuspension from 40 to 65° C. for from 30 min. to 72 hrs., a fumaraseactivity is lost, and microbial mass having theethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity can beprepared. By suspending the resulting microbial mass in aqueoussolutions containing fumaric or maleic acid and various amines to allowthem to react, aqueous solutions of optically activeS,S-diaminoalkylene-N,N′-disuccinates that do not contain by-productssuch as malic acid can be prepared.

Moreover, bacteriological properties of Brevundimonas diminuta strainMR-E001 are shown in the table below.

TABLE 1 Bacteriological properties of MR-E001 strain Morphologyrod-shaped Gram stain − Spores − Mobility + Flagella polar flagellationOxygen requirement aerobic Oxidase + Catalase + OF test − Color tone ofcolonies No characteristic pigment is generated. Production offluorescent pigment − Accumulation of PHB + Auxotrophy present Quinonesystem Q-10 Reduction of nitrates + Production of Indole − Argininedihydrolase − Urea degradation − Esculin degradation − Gelatinliquefaction − PNPG − Assimilation − Glucose L-Arabinose − D-Mannose −D-Mannitol − N-Acetyl-D-glucosamine − Maltose − Potassium gluconate +n-Capric acid − Adipic acid + dl-Malic acid + Citric acid + Phenylacetate −

Moreover, Brevundimonas diminuta strain MR-E001 was deposited on Feb. 5,2003, with International Patent Organism Depositary, National Instituteof Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan), under the Accession No. FERMBP-08677.

The present invention is described more in detail by referring to theExamples below. However, the Examples should be considered as beingpresented for further understanding of the present invention, and do notlimit the scope of the present invention by any means.

Example 1 Preparation of Wild TypeEthylenediamine-N,N′-Disuccinate:Ethylenediamine Lyase Gene Derived fromMR-E001 Strain

(1) Preparation of Chromosomal DNA from MR-E001 Strain

The MR-E001 strain was cultured with shaking in 100 ml of an EDDS medium[0.2% ethylenediamine-N,N′-disuccinate, 0.2% glucose, 0.1% Bacto yeastextract, 0.05% polypeptone, 0.1% MgSO₄.7H₂O, 25% (v/v) phosphate buffer(1M, pH 7.0), a 0.5% (v/v) mixture solution of metal salts (containing56 g of NaSO₄, 8 g of MgCl₂.6H₂O, 0.8 g of CaCl₂, 0.6 g of MnSO₄.4H₂O,0.12 g of FeCl₃.6H₂O, and 0.06 g of ZnSO₄ per 100 ml)] at 30° C. for 4days. Then, the cells were harvested and suspended in 4 ml of asaline-EDTA solution (0.1 M EDTA, 15 M NaCl, pH 8.0), to which 8 mg oflysozyme was added. The resulting suspension was shaken at 37° C. for 1hr. and then frozen. Next, 10 ml of a Tris-SDS solution (1% SDS, 0.1 MNaCl, 0.1 M Tris, pH 9.0) was gently added thereto while shaking, andfurther proteinase K (from Merck) was added (at the final concentrationof 1 mg) and shaken at 37° C. for 1 hr. Then, an equal volume ofTE-saturated phenol (TE:10 mM Tris, 1 mM EDTA, pH 8.0) was added theretoand stirred followed by centrifugation. The upper layer was collected,to which a two-fold volume of ethanol was added, and then DNA wascollected by rolling with a glass rod followed by removal of phenolusing 90%, 80%, and 70% ethanol in this order. Subsequently, the DNA wasdissolved in 3 ml of a TE buffer, to which a ribonuclease A solution(that has been treated by heat at 100° C. for 15 min.) was added at thefinal concentration of 10 mg/ml to shake at 37° C. for 30 min.Proteinase K was further added to shake at 37° C. for 30 min. Then, anequal volume of TE-saturated phenol was added thereto, which wasseparated by centrifugation into upper and lower layers to collect theupper layer (hereinafter, this procedure is referred to as phenolextraction). After phenol extraction was repeated twice, an equal volumeof chloroform (containing 4% isoamyl alcohol) was added to repeat thesimilar extraction procedure twice (hereinafter, this procedure isreferred to as chloroform extraction). Next, ethanol twice in volumethereof was added to the upper layer, and the DNA was recovered byrolling with a glass rod to obtain a chromosomal DNA sample.

(2) Preparation of Probe

The present applicants previously succeeded in isolating anethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene fromBrevundimonas sp. strain TN-3, and determined for the first time theamino acid sequence and the gene sequence thereof [JP Patent Publication(Kokai) No. 10-210984A (1998)]. The degenerate primers used in the abovepublication, i.e., artificial DNAs having the sequences represented bySEQ ID NO: 3 and SEQ ID NO: 4 (Primer #1 and Primer #2, respectively)were used, and PCR was conducted by using the chromosomal DNA from theMR-E001 strain obtained in step (1).

(SEQ ID NO: 3) Primer #1: ATGACICCIC AYAAYCCIGA YGC (SEQ ID NO: 4)Primer #2: CCDATYTGCAT YTTICCIGC RACIGAICCD ATYTC

Specifically, 1 μl of the chromosomal DNA from the MR-E001 strain, 10 μlof 10× buffer for the reaction, 4 μl of 10 mM dNTP, 1 μl of Primer #1and 1 μl of Primer #2 (equivalent to 100 pmol, respectively), and 1 μlof ExTaq (from Takara Shuzo) were admixed to obtain a 100 μl solution.The resulting solution was incubated at 95° C. for 30 sec.(denaturation), at 55° C. for 30 sec. (annealing), and at 72° C. for 2min. (extension) for 30 cycles. After the completion of the reaction,phenol extraction and chloroform extraction were performed to recoverthe amplified DNA by ethanol precipitation. The resulting DNA wasseparated by 1.0% agarose gel electrophoresis, and then obtained was aDNA fragment having about 300 bp, which was expected to encode a part ofthe ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene of theMR-E001 strain. This DNA fragment obtained thereby was labeled by usinga DIG DNA Labeling kit (from Roche Diagnostics) to provide a probe.

(3) Preparation of DNA Library

To 10 μl of the chromosomal DNA from the MR-E001 strain, 5 μl of 10×buffer for restriction digestion, 33 μl of sterile water, and 2 μl of arestriction enzyme KpnI were added to react at 37° C. for 16 hrs., andthen the DNA was recovered by ethanol precipitation. DNA fragments from6.5 Kb to 5.5 Kb were extracted by agarose gel electrophoresis from thegel, which were recovered by using a DNA PREP (from Diatron). These DNAfragments were inserted at the KpnI site of an E. coli vector pUC18 byusing a DNA Ligation Kit Ver. 1 (from Takara Shuzo) to prepare arecombinant DNA library. The pUC18 fragment used for the ligation wasprepared by the process described below. To 2 μl of a solutionpreserving pUC18, μl of 10× buffer for restriction enzyme, 40 μl ofsterile water, and 3 μl of a restriction enzyme KpnI were added to reactat 37° C. for 2 hrs., and then phenol extraction and chloroformextraction were performed followed by ethanol precipitation. Theprecipitate was dried, which then was dissolved into 50 μl of sterilewater. Further, thereto, 1 μl of alkaline phosphatase (from TakaraShuzo), 10 μl of 10× buffer, and 39 μl of sterile water to react at 65°C., and then phenol extraction and chloroform extraction were performedfollowed by ethanol precipitation. The precipitate was dried, which thenwas dissolved into sterile water.

(4) Preparation of E. Coli Transformants and Selection of RecombinantDNA

E. coli strain JM109 was inoculated into 1 ml of an LB medium (1% Bactotryptone, 0.5% Bacto yeast extract, 0.5% NaCl) to pre-culture at 37° C.for 5 hrs. under aerobic conditions. To 40 ml of an SOB medium (2% Bactotryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO₄,1 mM MgCl₂), 0.4 ml of this culture was added to culture at 18° C. for20 hrs. From this culture, cells were harvested by centrifugation, towhich then 13 ml of a cool TF solution [20 mM PIPES-KOH (pH 6.0), 200 mMKCl, 10 mM CaCl₂, 40 mM MnCl₂] was added to stand at 0° C. for 10 min.followed by the second centrifugation. After the supernatant wasremoved, the precipitated E. coli was suspended in 3.2 ml of a cool TFsolution, and 0.22 ml of dimethyl sulfoxide was added thereto to standat 0° C. for 10 min. To 200 μl of the competent cells prepared thereby,10 μl of the solution containing the recombinant plasmid DNA (the DNAlibrary) prepared in step (3) was added to stand at 0° C. for 30 min.,to which then heat shock was given at 42° C. for 30 sec. The cells werecooled at 0° C. for 2 min., to which 1 ml of an SOC medium (20 mMglucose, 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mMKCl, 1 mM MgSO₄, 1 mM MgCl₂) was added to culture with shaking at 37° C.for 1 hr. The resulting culture was divided into 200 μl aliquots, eachof which was inoculated onto a LBamp medium (a LB medium containing 100mg/l ampicillin and 1.5% agar) to culture at 37° C. From the colonies oftransformants grown on the agar medium, transformants expected to carrythe ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene wereselected by colony hybridization. Specifically, the transformants grownon the agar medium were transferred on a nylon membrane (Biodyne A, fromNihon Pall), where microbial cells were lysed, and DNA was fixed. Thefixed DNA was treated with the probe (about 300 bp) prepared in step (2)to select colonies having the recombinant DNA of interest by using a DIGLuminescent Detection Kit (from Roche Diagnostics).

(5) Preparation of Recombinant Plasmid

The transformant selected in step (4) was cultured in 100 ml of an LBAmpmedium (an LB medium containing 100 mg/L ampicillin) at 37° C.overnight. After cells were harvested, the plasmid DNA was recovered byusing a Flexi Prep (from Amersham Biosciences). The resultingrecombinant plasmid DNA was named pEDS9001.

(6) Preparation of Restriction Map and Definition ofEthylenediamine-N,N′-Disuccinate:Ethylenediamine Lyase Gene Region

The plasmid pEDS9001 obtained in step (5) was cleaved by severalrestriction enzymes to make a restriction map (FIG. 1). In addition, theplasmid was subcloned by an ordinary method. Specifically, afterpEDS9001 was cleaved by a restriction enzyme BamHI, agarose gelelectrophoresis was performed to extract from the gel a DNA fragment ofabout 5.3 Kb, which was recovered by using a DNA PREP (from Diatron).After autoligation by using a DNA Ligation Kit Ver. 1 (from TakaraShuzo), E. coli strain JM109 was transformed to obtain a plasmid(pEDS9003) (FIG. 2), wherein a fragment of about 2.6 Kb expected tocontain the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase geneis inserted.

(7) Determination of Nucleotide Sequence

A nucleotide sequence around the region defined in step (6) wasdetermined by using a fluorescence sequencer ALFII (from AmershamBiosciences). As a result, the nucleotide sequence (SEQ ID NO: 2)encoding an open reading frame made of the amino acid sequence of SEQ IDNO: 1 was discovered.

Example 2 Evaluation of Activity of Transformant Wherein Wild TypeEthylenediamine-N,N′-Disuccinate:Ethylenediamine Lyase Gene Derived fromMR-E001 Strain is Transferred

To 2 μl of the recombinant plasmid pEDS9003 having theethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene obtained instep (6) of Example 1, 2 μl of 10× buffer for restriction enzyme, 15 μlof sterile water, and 1 μl of the restriction enzyme KpnI were added toreact at 37° C. for 2 hrs., and then plasmid DNA was recovered byethanol precipitation. The plasmid DNA was dried, which then wasdissolved into 17 μl of sterile water, 2 μl of 10× buffer forrestriction enzyme, and 1 μl of the restriction enzyme BamHI were addedto react at 37° C. for 2 hrs. From this reaction solution, a fragment ofabout 2.6 Kb is prepared by agarose gel electrophoresis, which wasinserted into an E. coli vector pUC 119. E. coli strain JM109 wastransformed by using the prepared ligation solution to obtain a plasmidof interest. The thus prepared plasmid was named pEDS9020 (FIG. 3), andthe transformant was named JM109/pEDS9020. Each of JM109/pEDS9020 andJM109/pEDS020 [described in JP Patent Publication (Kokai) No. 10-210984A(1998)] as a control was inoculated into 1 ml of an LBAmp medium toculture with shaking at 37° C. for 8 hrs., and then cultured in 40 ml ofan LBAmp medium containing 1 mM isopropyl-β-thiogalactoside at 37° C.for 30 hrs. The resulting culture was washed with a 10 mM sodiumphosphate buffer solution (pH 8.0), and then was suspended in 2 ml ofthe buffer solution. A part of the resulting cell suspension wassuspended in 50 ml of an aqueous solution, pH of which was 8.0,containing 342 mM fumaric acid and 171 mM ethylenediamine to react at30° C. A part (0.1 ml) of the reaction mixture was sampled at intervals,and was added into 0.9 ml of a 0.42 N NaOH aqueous solution to stop thereaction. After the cells were removed by centrifugation,S,S-ethylenediamine-N,N′-disuccinate generated thereby was analyzed byusing HPLC {WAKOSIL5C8 (from Wako Pure Chemical) [the eluate: 50 mMphosphoric acid containing 10 mM tetra-n-butyl ammonium hydroxide and0.4 mM CuSO₄; pH2]}. One enzyme unit (U) was defined as the amount ofenzyme to produce 1 μmol of S,S-ethylenediamine-N,N′-disuccinate perminute under the above determination conditions. The activities ofJM109/pEDS9020 and JM109/pEDS020 per cell (per OD630) were determined tobe 1.22 mU/ODml and 0.89 mU/ODml, respectively. It was confirmed thatthe ethylenediamine-N,N′-disuccinate:ethylenediamine lyase derived fromthe MR-E001 strain has a high activity of the enzyme. Moreover, pEDS9020was deposited on Feb. 5, 2003, with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan),under the Accession No. FERM BP-08676.

Example 3 Preparation of ModifiedEthylenediamine-N,N′-Disuccinate:Ethylenediamine Lyase Genes

(1) Induction of Mutations into the Wild TypeEthylenediamine-N,N′-Disuccinate:Ethylenediamine Lyase Gene

By using the plasmid pEDS9020 obtained in Example 2, mutations wereinduced on a random basis into the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene. Forinducing mutations, utilized was base substitution by nucleotidemisincorporation by PCR. The oligonucleotide ED-01 (SEQ ID NO: 5), whichcontains the initiation codon region of theethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene, and theoligonucleotide ED-02 (SEQ ID NO: 6), which contains the downstreamregion by about 50 bp from the termination codon of the gene, wereprimers for PCR inducing mutations, and 100 μl of a PCR reactionsolution having a composition described below was prepared:

Primer:

(SEQ ID NO: 5) ED-01: CGCCATGGCC CCGCATAACC CAGATGCCAC C(The underlined part is a cleavage recognition site by the restrictionenzyme NcoI); and

(SEQ ID NO: 6) ED-02: AAACAAGCTT CGTCATGGCT ATCCCCTC(The underlined part is a cleavage recognition site by the restrictionenzyme HindIII).

Composition of Reaction Solution:

Template DNA (pEDS9020 prepared in the above step) 1 μl

10×PCR buffer (from GIBCO) 10 μl

50 mM MgCl₂ (from GIBCO) 3 μl

Primer ED-01 1 μl

Primer ED-02 1 μl

2.5 mM dNTP 2 μl each

10 mM dITP 2 μl

10 mM dBraUTP 2 μl

Sterile water 71 μl

Taq DNA polymerase (from GIBCO) 1 μl

The above reaction solution was incubated at 94° C. for 30 sec.(denaturation), and at 68° C. for 180 sec. (annealing and extension) for30 cycles. After the above PCR was completed, an amplified fragment ofabout 1.5 kb was detected from 10 μl of the reaction solution by using0.7% agarose gel electrophoresis. In addition, the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene wasamplified by usual PCR to use it as a control for evaluating heatresistance. Specifically, 100 μl of a PCR reaction solution having acomposition described below was prepared:

Template DNA (pEDS9020) 1 μl

10× Pyrobest Buffer (from Takara Shuzo) 10 μl

Primer ED-01 1 μl

Primer ED-02 1 μl

5 mM dNTP 2 μl each

Sterile water 78 μl

Pyrobest™ DNA polymerase (from Takara Shuzo) 1 μl

The above reaction solution was incubated at 94° C. for 30 sec.(denaturation), and at 68° C. for 180 sec. (annealing and extension) for30 cycles. After the above PCR was completed, an amplified fragment ofabout 1.5 kb was detected from 10 μl of the reaction solution by using0.7% agarose gel electrophoresis.

In Primer ED-01 and Primer ED-02, the cleavage recognition site by therestriction enzyme NcoI, and the cleavage recognition site by therestriction enzyme HindIII were transferred, respectively (theunderlined parts of the nucleotide sequences of Primer ED-01 and PrimerED-02). The amplified DNA product can be readily inserted, by thecleavage thereof using both of the restriction enzymes, into between theNcoI site and the HindIII of an expression vector pFY529V, which will bedescribed later.

(2) Construction of Expression Vector

In order to efficiently detect a remainingethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity afterheat treatment in a screening step, which will be described later, an E.coli expression vector pFY529V, which has a high copy number and greatexpression efficiency, was prepared (FIG. 4). To 5 μl of an expressionvector pKK233-2 having a trc promoter (from Amersham), 1 μl of arestriction enzyme NaeI, 1 μl of a restriction enzyme ScaI, 1 μl of 10×buffer for restriction digestion, and 2 μl of sterile water were addedto perform cleavage reaction at 37° C. for 12 hrs. After the cleavage,an NaeI-ScaI fragment (1.2 kb) that does not contain the replicationorigin of the plasmid was extracted by 0.7% agarose gel electrophoresis,and 3 μl of a TE solution (10 mM Tris, 1 mM EDTA, pH 8.0) containing theDNA fragment was recovered by using a DNA PREP (from Diatron). In tandemwith this procedure, to 2 μl of a vector pUC18 having a high copynumber, 1 μl of a restriction enzyme PvuII, 1 μl of a restriction enzymeScaI, 1 μl of 10× buffer for restriction digestion, and 5 μl of sterilewater were added to perform cleavage reaction at 37° C. for 12 hrs.After the cleavage, a PvuII-ScaI fragment (1.6 kb) that contains thereplication origin of the plasmid was extracted by 0.7% agarose gelelectrophoresis, and 1 μl of a TE solution containing the DNA fragmentwas recovered by using a DNA PREP (from Diatron). Both of the resultingDNA fragments were ligated by using a DNA Ligation Kit Ver. 1 (fromTakara Shuzo). By admixing 3 μl of a solution of the NaeI-ScaI fragmentfrom pKK233-2, 1 μl of a solution of the PvuII-ScaI fragment from pUC18,16 μl of an A solution of the kit, and 4 μl of a B solution of the kit,the ligation was performed at 16° C. for 16 hrs. By using a reactionsolution after the ligation, E. coli JM109 strain was transformed by themethod described in Example 1 (4). Inoculated was about 10 clones fromthe resulting transformant colony into 1.5 ml of an LBAmp medium, andincubated with shaking at 37° C. for 12 hrs. After the incubation, cellswere harvested from the culture by centrifugation followed by theextraction of the plasmid DNA by using a Flexi Prep (from AmershamBiosciences). After the resulting plasmid DNA was cleaved by therestriction enzyme ScaI, a clone wherein the NaeI-ScaI fragment (1.2 kb)from pKK233-2 and the PvuII-ScaI fragment (1.6 kb) from pUC18 werecorrectly ligated was selected by 0.7% agarose gel electrophoresis,which was named pFY529V, to use as an expression vector for a mutationlibrary.

(3) Preparation of Mutation Library

A reaction solution containing theethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes wherein themutations were induced that was obtained by PCR in step (1) was purifiedby ethanol precipitation according to the conventional method, and aprecipitate was again suspended in 70 μl of sterile water. Thereto, 10μl of 10× buffer for restriction digestion, 10 μl of a restrictionenzyme NcoI, and 10 μl of a restriction enzyme HindIII were added toperform cleavage reaction at 37° C. for 12 hrs. After the cleavagereaction, phenol extraction and chloroform extraction were conductedfollowed by ethanol precipitation. The precipitate was again suspendedin 100 μl of sterile water to obtain a mutated DNA fragment solution. Intandem with this procedure, to 3 μl of the expression vector pFY529Vprepared in step (2), 67 μl of sterile water, 10 μl of 10× buffer forrestriction digestion, 10 μl of a restriction enzyme NcoI, and 10 μl ofa restriction enzyme HindIII were added to perform cleavage reaction at37° C. for 12 hrs. After the cleavage, phenol extraction and chloroformextraction were conducted followed by ethanol precipitation forpurification. Then, the precipitate was again suspended in 10 μl ofsterile water to obtain a cleaved pFY529V solution. The mutated DNAfragments and the expression vector pFY529V were ligated by using a DNALigation Kit Ver. 1 (from Takara Shuzo). By admixing 3 μl of the abovemutated DNA fragment solution, 1 μl of the above cleaved pFY529Vsolution, 16 μl of an A solution of the kit, and 4 μl of a B solution ofthe kit, the ligation was performed at 16° C. for 16 hrs. By using areaction solution after the ligation, E. coli JM109 strain wastransformed by the method described in Example 1 [step (4)] to obtaintransformants bearing various mutation induced EDDSase genes. Inaddition, the similar procedure was performed wherein the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene that wasamplified in step (1) was used as a fragment for insertion. A plasmidwas extracted from the resulting transformant colony, and a nucleotidesequence thereof was confirmed by using a fluorescence sequencer ALF II(from Amersham Biosciences). The result was identical with thenucleotide sequence of the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase (SEQ ID NO: 2)determined in Example 1 [step (7)] {besides the change at the fourthbase (A→G) due to the transferred NcoI site in Primer ED-01}. Thisplasmid was named pEDTrc9003, and E. coli containing the plasmid, i.e.,JM109/pEDTrc9003, was used as a control in step (4) of screening for anethylenediamine-N,N′-disuccinate:ethylenediamine lyase with improvedheat resistance, which will be described later. Moreover, pEDTrc9003 wasdeposited on Feb. 5, 2003, with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan),under the Accession No. FERM BP-08675.

(4) Screening for Ethylenediamine-N,N′-Disuccinate:Ethylenediamine Lyasewith Improved Heat Resistance

The JM109 transformants containing the mutatedethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes obtained instep (3), and JM109/pEDTrc9003 as a control were inoculated into anLBAmp medium, which had been dispensed in a 48-hole multi-dish in a 1.5ml aliquot, for liquid culture at 37° C. for 12 hrs. The resultingculture was treated by heat at 50° C. for 30 min. followed by thedetermination of remainingethylenediamine-N,N′-disuccinate:ethylenediamine lyase activityaccording to the method described in Example 2. About 10,000 strains ofthe transformants obtained in step (3) were screened. As a result, fourstrains wherein the enzyme activity remained were obtained, whereas theactivity was already completely lost in JM109/pEDTrc9003 bearing thewild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase.

(5) Identification of Mutations

The ethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes whereinthe mutations were induced contained in the four candidate strains withimproved heat resistance that were obtained in step (4) were analyzed inorder to confirm what type of and where the mutation was inducedaccording to the procedure described below. Recombinant plasmid DNAscontained in the four candidate strains with improved heat resistancewere purified by using a Flexi Prep (from Amersham Biosciences), and theresulting recombinant plasmid DNAs were named pEDTrcI-2, pEDTrcI-23,pEDTrcJ-05, and pEDTrcK-01, respectively. In addition, mutated enzymesthemselves contained in the respective plasmids were named I-2, I-23,J-05, and K-01. Nucleotide sequences of the mutatedethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes containedin these recombinant plasmid DNAs were determined by using afluorescence sequencer ALF II (from Amersham Biosciences). When thedetermined nucleotide sequence of each of the mutatedethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes and thenucleotide sequence of the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase (SEQ ID NO: 2)were compared, it was shown that in pEDTrcI-2, the isoleucine residue(ATC) at 166 was substituted with threonine (ACC); in pEDTrcI-23, thelysine residue (AAA) at 120 was substituted with glutamic acid (GAA); inpEDTrcJ-05, the isoleucine residue (ATC) at 166 was substituted withserine (AGC); and in pEDTrcK-01, the alanine residue (GCC) at 365 wassubstituted with valine (GTC), each of the recombinant plasmid DNAsbeing a single mutant [the change in nucleotide sequence encoding eachof the amino acids is indicated in (---)].

(6) Evaluation of Heat Resistance of Single Mutants

Heat resistance of the four single mutants of theethylenediamine-N,N′-disuccinate:ethylenediamine lyase (I-2, I-23, J-05,and K-01), which were identified in step (5), was evaluated morethoroughly. Four E. coli strains bearing the single mutants of theethylenediamine-N,N′-disuccinate:ethylenediamine lyase (JM109/pEDTrcI-2,JM109/pEDTrcI-23, JM109/pEDTrcJ-05, and JM109/pEDTrck-01), and E. colibearing the wild type ethylenediamine-N,N′-disuccinate:ethylenediaminelyase JM109/pEDTrc9003 were inoculated into 1.5 ml of an LBAmp medium toincubate with shaking at 30° C. for 8 hrs. Then, 400 μl of each of theculture solutions was inoculated into 40 ml of an LBAmp medium preparedin a 500 ml Erlenmeyer's flask to culture with shaking at 37° C. for 12hrs. Collected was 1.5 ml of the resulting culture and cells wereharvested by centrifugation followed by washing with a 50 mM boric acidbuffer solution (pH 9.0). Then, the cells were suspended in 1.5 ml ofthe buffer solution to prepare a cell suspension. This cell suspensionwas sonicated to destruct the cells and obtain crude enzyme extract. Theresulting crude extract was subjected to heat treatment for 30 min. at40, 45, 50, 55, 60, and 65° C., and then immediately cooled to 4° C.After the heat treatment, theethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity wasmeasured according to the method described in Example 2. For eachmutant, the relative remaining activity to the control (100%), which waskept cooled at 4° C. without heat treatment, was determined

The results are shown in FIG. 5. In FIG. 5, the vertical axis representsa remaining ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseactivity, and the horizontal axis represents a treatment temperature.The wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyasecompletely lost its enzyme activity at 50° C. On the other hand, thesingle mutants, I-2, I-23, J-05, and K-01, retained the relativeremaining activities of 75% at 50° C., 85% at 50° C., 12% at 60° C., and12% at 55° C., respectively, and it was confirmed that the heatresistance of these single mutants improved compared with that of thewild type.

Example 4 Preparation of Multiple Mutants and Evaluation of HeatResistance Thereof (1) Preparation of Multiple Mutants

Multiple mutants were prepared by combining the amino acid substitutionsin the four single mutants whose improved heat resistance was confirmedin Example 3. The mutation sites of the single mutants, I-2, I-23, J-05,and K-01, were the isoleucine residue at 166, the lysine residue at 120,the isoleucine residue at 166, and the alanine residue at 366,respectively. By cleaving theethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNA fragmentby restriction enzymes EcoT22I, AccI, and ClaI, provided were DNAfragments, each of which contains one of these mutation sites (FIG. 6).By allowing the cleaved DNA fragment of a single mutant to replace thecorresponding DNA fragment derived from a different single mutant andreligate it, a chimeric enzyme gene having double mutation or triplemutation was formed (FIG. 6). For example, for preparing a doublemutant, Ch-4, first, pEDTrcI-23 that contained a gene encoding thesingle mutant I-23 was cleaved by restriction enzymes NcoI and AccI, andthe shorter fragment (about 0.5 kb) of the resulting DNA fragments wasextracted by agarose gel electrophoresis. In tandem with this procedure,pEDTrcJ-05 that contained a gene encoding the single mutant J-05 wascleaved by restriction enzymes NcoI and AccI, and the longer fragment(about 3.8 kb including the vector) of the resulting DNA fragments wasextracted by agarose gel electrophoresis. Both of the DNA fragments wererecovered by using a DNA PREP (from Diatron) and ligated by using a DNALigation Kit Ver. 1 (from Takara Shuzo), and then E. coli JM109 wastransformed therewith according to the conventional method. From theresulting transformant, a plasmid was extracted and cleaved byrestriction enzymes NcoI and AccI to confirm that the ligation wascorrect by agarose gel electrophoresis. This plasmid DNA and thechimeric enzyme were named pEDTrcCh-4 and Ch-4, respectively. Likewise,an NcoI-AccI fragment (about 0.5 kb) from the single mutant I-23 and anNcoI-AccI fragment (about 3.8 kb including the vector) from the singlemutant I-2 were ligated to prepare plasmid DNA pEDTrcCh-6 and a chimericenzyme Ch-6. According to the similar procedure, an NcoI-ClaI fragment(about 1 kb) from the single mutant J-05 and an NcoI-ClaI fragment(about 3.3 kb including the vector) from the single mutant K-01 wereligated to prepare plasmid DNA pEDTrcCh-1 and a chimeric enzyme Ch-1. AnNcoI-ClaI fragment (about 1 kb) from the single mutant I-2 and anNcoI-ClaI fragment (about 3.3 kb including the vector) from the singlemutant K-01 were ligated to prepare plasmid DNA pEDTrcCh-2 and achimeric enzyme Ch-2. Further, for preparing triple mutants, anNcoI-AccI fragment (about 0.5 kb) from the single mutant I-23 and anAccI-ClaI fragment (about 0.5 kb) from the single mutant J-05, and anNcoI-ClaI fragment (about 3.3 kb including the vector) from the singlemutant K-01 are ligated to prepare plasmid DNA pEDTrcCh-8 and a chimericenzyme Ch-8. An NcoI-AccI fragment (about 0.5 kb) from the single mutantI-23 and an AccI-ClaI fragment (about 0.5 kb) from the single mutantI-2, and an NcoI-ClaI fragment (about 3.3 kb including the vector) fromthe single mutant K-01 are ligated to prepare plasmid DNA pEDTrcCh-10and a chimeric enzyme Ch-10. The structures of the resulting chimericenzymes are shown in FIG. 6.

(2) Evaluation of Heat Resistance of Multiple Mutants

Heat resistance of the six multiple mutants of theethylenediamine-N,N′-disuccinate:ethylenediamine lyase (Ch-4, Ch-6,Ch-1, Ch-2, Ch-8, and Ch-10), which were prepared in step (1), wasevaluated. Six E. coli strains bearing the multiple mutants of theethylenediamine-N,N′-disuccinate:ethylenediamine lyase(JM109/pEDTrcCh-4, JM109/pEDTrcCh-6, JM109/pEDTrcCh-1, JM109/pEDTrcCh-2,JM109/pEDTrcCh-8, and JM109/pEDTrcCh-10), and E. coli bearing the wildtype ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseJM109/pEDTrc9003 were inoculated into 1.5 ml of an LBAmp medium toincubate with shaking at 30° C. for 8 hrs. Then, 400 μl of each of theculture solutions was inoculated into 40 ml of an LBAmp medium preparedin a 500 ml Erlenmeyer's flask to culture with shaking at 37° C. for 12hrs. Collected was 1.5 ml of the resulting culture and cells wereharvested by centrifugation followed by washing with a 50 mM boric acidbuffer solution (pH 9.0). Then, the cells were suspended in 1.5 ml ofthe buffer solution to prepare a cell suspension. This cell suspensionwas sonicated to destruct the cells and obtain a crude enzyme extract.The resulting crude extract was subjected to heat treatment for 30 min.at 40, 45, 50, 55, 60, and 65° C., and then immediately cooled to 4° C.After the heat treatment, theethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity wasmeasured according to the method described in Example 2. For eachmutant, the relative remaining activity to the control, which was keptcooled at 4° C. without heat treatment, was determined. The results areshown in FIG. 7. In FIG. 7, the vertical axis represents a remainingethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity, and thehorizontal axis represents a treatment temperature. The wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase completely lostits enzyme activity at 50° C. On the other hand, the multiple mutants,Ch-4, Ch-6, Ch-1, Ch-2, Ch-8, and Ch-10, retained the relative remainingactivities of 72% at 60° C., 89% at 55° C., 56% at 60° C., 25% at 55°C., 90% at 60° C., and 97% at 55° C., respectively, and it was confirmedthat the heat resistance of these multiple mutants improved comparedwith that of the wild type and the single mutants.

All references, articles, publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entireties.

INDUSTRIAL APPLICABILITY

The present invention provides a nucleotide sequence and an amino acidsequence of a wild type ethylenediamine-N,N′-disuccinate:ethylenediaminelyase derived from Brevundimonas diminuta strain MR-E001. Further, thepresent invention provides a nucleotide sequence and an amino acidsequence of a modified ethylenediamine-N,N′-disuccinate:ethylenediaminelyase derived from the wild typeethylenediamine-N,N′-disuccinate:ethylenediamine lyase. Moreover, thepresent invention provides recombinant DNAs containing the wild type andthe modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyasegenes; transformants or transductants containing the recombinant DNAs;and a method of preparing diaminoalkylene-N,N′-disuccinates by using thetransformants or transductants. By the present invention,diaminoalkylene-N,N′-disuccinates can be prepared efficiently.

Sequence Table Free Text

SEQ ID NO: 1: Xaa represents Met or deletion

SEQ ID NO: 3: Artificial DNA

SEQ ID NO: 4: Artificial DNA

SEQ ID NO: 5: Artificial DNA

SEQ ID NO: 6: Artificial DNA

1-53. (canceled)
 54. A method of producing adiaminoalkylene-N,N′-disuccinate, comprising: reacting fumaric acid anda diamine in the presence of a transformant or transductant; andcollecting the diaminoalkylene-N,N′-disuccinate resulting from thereaction products. wherein the transformant or transductant comprises arecombinant DNA wherein a gene encoding a protein comprising the aminoacid sequence of SEQ ID NO: 1 is inserted into a vector DNA, and theprotein has ethylenediamine-N,N′-disuccinate:ethylenediamine lyaseactivity.